O-GlcNAc转移酶Ogt在抗病毒天然免疫反应中的作用及其机制研究

Innate immune responses triggered by pattern-recognition receptors (PRRs) play an important role in immune defenses of host recognizing and defending pathogen-associated molecular patterns (PAMPs). However, inappropriate activation of PRRs can lead to prolonged inflammation and even to autoimmune and inflammatory diseases. Thus, PRR-triggered responses are regulated through many of the numerous distinct mechanisms for the cross-regulation of innate immune receptor signaling. A reciprocal interaction between TLR signaling and metabolic pathways has drawn great attention in recent years. O-GlcNAcylation plays an important role in multiple biological process. However, little attention was paid to the function of O-GlcNAcylation in immune system. Whether O-GlcNAc transferase (Ogt) can regulate virus-triggered immune response remains unknown. Our results showed that O-GlcNAcylation of proteins significantly decreased in Ogt-deficient macrophages. RNAi knockdown of Ogt significantly suppressed RNA virus (VSV、SeV) and poly (I:C) induced production of type I IFN (IFN-α, IFN-β) in macrophages. And, RNA virus (VSV、SeV) and poly (I:C) induced mRNA level of type I IFN (IFN-α, IFN-β) were significantly reduced in Ogt-deficient peritoneal macrophages. Furthermore, over-expression of Ogt significantly promoted RIG-I、MAVS-activated expression of IFN-β reporter gene. In addition, deficiency of Ogt barely affected MAPK, NF-κB or IRF3 signaling pathways in macrophages infected with VSV. These results indicated that Ogt is involved in the positive regulation of innate anti-viral immune response. Furthermore, O-GlcNAcylation of IRF3 significantly decreased in Ogt-deficient macrophages. The primary aim of this study is to investigate the regulation of virus-triggered innate anti-viral immune response by Ogt in macrophages as well as the underlying mechanisms, explore the function of O-GlcNAcylation in innate anti-viral immune response, enrich the regulation network of innate immunity and shed new light on the treatment of infectious diseases.

近年来代谢途径与PRR信号的交叉调控受到了广泛关注。糖代谢相关O-GlcNAc 修饰在多个生物学过程中发挥重要作用，而在免疫系统中的功能尚不清楚。我们前期研究发现巨噬细胞中大量蛋白存在着受Ogt调控的O-GlcNAc修饰，并且在Ogt条件性敲除的巨噬细胞中VSV、SeV 或poly (I:C) 诱导的I 型干扰素水平明显降低，但其胞浆信号通路却几乎不受影响，提示Ogt在 I 型干扰素的产生中发挥正向调控作用。并且初步机制探讨发现IRF3存在着O-GlcNAc修饰且在Ogt条件性敲除的巨噬细胞中明显降低，本项目拟进一步探讨Ogt在抗病毒天然免疫中的调控作用；寻找Ogt的靶分子及明确靶分子的O-GlcNAc修饰是否参与调控抗病毒天然免疫应答。本项目旨在揭示Ogt对抗病毒天然免疫应答的调控及其机制，阐明O-GlcNAc 修饰在抗病毒天然免疫应答中的作用，为感染性疾病的治疗提供潜在方向。

Ogt作为O-GlcNAc糖基化修饰的关键酶在多个生物学过程中发挥重要作用，而在免疫系统中的功能尚不清楚。本研究发现腹腔巨噬细胞中存在着大量O-GlcNAc糖基化修饰的蛋白，并且当巨噬细胞特异性敲除Ogt的表达后O-GlcNAc糖基化修饰水平明显降低。进一步通过利用Ogt抑制剂OSMI-1或者HBP糖代谢通路抑制剂DON，发现Ogt抑制剂OSMI-1或者HBP糖代谢通路抑制剂DON均可以明显抑制细胞因子IFN-α和IFN-β的产生。这说明巨噬细胞中存在受Ogt调控的O-GlcNAc糖基化修饰的蛋白并且O-GlcNAc糖基化修饰影响巨噬细胞中细胞因子的产生。并且进一步通过Ogt特异性siRNA和Ogt巨噬细胞条件性敲除小鼠特异性敲低或敲除巨噬细胞中Ogt的表达，发现Ogt特异性siRNA或Ogt条件性敲除的巨噬细胞中RNA病毒诱导的I型干扰素水平降低，过表达Ogt可以促进MAVS活化的IFN-β报告基因活化，提示Ogt可以正向调控I型干扰素的产生。进一步机制研究发现Ogt可以与抗病毒信号通路关键激酶IRF3的C末端383-419区域发生相互作用，并且IRF3的388、390、394、396、397可能是其发生O-GlcNAc修饰的潜在位点。并且IRF3的O-GlcNAc修饰可以通过促进IRF3的磷酸化水平和二聚化促进IRF3的活化水平，从而促进RNA病毒诱导的抗病毒细胞因子的产生。本研究揭示了Ogt对抗病毒天然免疫应答的调控及其机制，阐明O-GlcNAc糖基化修饰在抗病毒天然免疫应答中的作用，为感染性疾病的治疗提供潜在方向。